Age-Dependent Remodeling of Splenic Immune Responses in Murine Sepsis Revealed by Single-Cell Transcriptomics

Authors:
Christine Rodhouse, Dayuan Wang, Hongru Tang, Miguel Hernandez-Rios, Whitman Wiggins, Xuanxuan Yu, Leandro Balzano-Nogueira, Angel Charles, Larissa Langhi Prata, Tyler Loftus, Letitia Bible, Alicia Mohr, Robert Maile, Guoshuai Cai, Shawn Larson, Philip Efron, Jaimar Rincon

Body of Abstract:
Background: Decades of sepsis research have yet to produce an effective immune-therapeutic to improve patient outcomes. This is in part due to a failure to apply precision medicine to sepsis research, which includes accounting for the age of the host as the disease tends to affect the very young and older adults the most. Our goal is to delineate the transcriptomic patterns of leukocytes early after sepsis in key age (neonate, young adult, older adult) groups using a polymicrobial model of murine abdominal sepsis, allowing for comparisons of similarities and differences.

Methods: Neonatal (7-day-old), young adult (3-4 months), and older adult (18-24 months) C57BL/6 (B6) mice were challenged with 1.1 – 1.3 mg/g BW of cecal slurry (CS) to induce polymicrobial sepsis. Spleens were harvested prior to challenge (naive) and 18 hours post-sepsis (CS18h). Single-cell gene expression libraries were generated using the 10X Genomics platform and sequenced on an Illumina HiSeq® system (scRNAseq). Data was processed with CellRanger and analyzed in Seurat. Differentially expressed genes (DEGs) were identified using an adjusted Wilcoxon’s rank-sum test. IPA was used for functional enrichment, with significance assessed by Fisher’s exact test. Pseudotime trajectory analysis was inferred based on transcriptional dynamics.

Results: Following sepsis, neonates showed expansion of HSPCs, PMNs, and mast cells; young adults exhibited reductions in HSPCs and mast cells, while older adults displayed increased DCs with otherwise stable myeloid proportions. Monocyte transcriptional responses differed markedly by age. Neonatal monocytes showed dysregulated activation and impaired maturation with induction of IL-10, Th2, TLR, LPS/IL-1, iNOS, IL-17, and IL-1 pathways and suppression of LXR/RXR and MAPK. Young adults mounted a balanced response, upregulating IL-10, IL-17, and PD-1/PD-L1 while downregulating Th1, HGF, and IL-4. Aged monocytes exhibited profound dysregulation with activation of cytokine-storm, IL-10, ID3, and sirtuin pathways and loss of PPAR/RXR, TRIM21, and communication signaling, reflecting a hyperinflammatory, metabolically compromised state. Pseudotime trajectory analysis revealed pronounced alterations suggesting perturbations in hematopoietic differentiation dynamics across the ages. In young adult mice, myeloid differentiation skewed towards inflammatory effector cells (e.g. monocytes, DCs). In contrast, in neonates and aged mice there were disrupted trajectories and reduced progression to the terminal macrophage branches, indicative of impaired myeloid maturation.

Conclusions: scRNAseq analysis of splenic leukocytes revealed that host age results in tremendous differences in cell proportions, DEGs, and cell differentiation early after sepsis. Translation efforts in sepsis can be improved by applying precision medicine to research, including preclinical rodent sepsis models needed for FDA approval of therapies.